Antimicrobial peptides, their variants and uses

ABSTRACT

The present invention relates to novel antimicrobial peptide and variants thereof. The invention further relates to method of killing or inhibiting growth of microbes and use of the peptide here disclosed as a medicament, feed additive, preservative or surfactant.

PRIORITY

This application is a U.S national application of the internationalapplication number PCT/FI2016/050483 filed on Jun. 29, 2016 and claimingpriority of Finnish national application FI2015517 filed on Jun. 30,2015, the contents of both of which are incorporated herein byreference.

SEQUENCE LISTING

This application contains sequence listing provided on a written formatand as computer readable format which is identical to the written formatand is incorporated by reference in its entirety.

Sequence Listing with the specific file nameUpdated_sequence_listing_to_Patent_Office_PI100446.txt, having aparticular size of 20.7 kb, submitted to the PTO on Jun. 12, 2019, ishereby incorporated by reference in its full entirety and in toto.

FIELD OF INVENTION

The present invention relates to antimicrobial peptides or variantsthereof, to a method of killing or inhibiting growth of microbes and toa use of the peptide described here as a medicament, feed additive,preservative or surfactant.

BACKGROUND

Among drug compounds, antibiotics have had a tremendous impact on thelife expectancy of mankind. However, the capacity of microbes to developresistance is almost unlimited. Multi-drug resistance has become a verycommon and dangerous characteristic of many human pathogens, and drugresistance is spreading across the globe.

Endophytes are promising producers of a wide array of secondarymetabolites with potential application in biomedicine, pharmaceuticaland healthcare industries. The methodology typically used for studyingfunctional diversity of endophytes is based on isolation with anemphasis towards fast-growing strains, thus not representing the fullbiodiversity. Methods such as denaturing gradient gel electrophoresis(DGGE), restriction fragment length polymorphism (RFLP) or cloning anddirect sequencing are used for analyzing the diversity of unculturablefungal communities in plants, but such methods are not suitable for thefunctional studies. A frequent problem with endophytes is that theyproduce metabolites only for a certain period of time in vitro and thenbecome inactive, lose viability or ability to produce bioactivesecondary metabolites. Such culturing problems on other microbes haveearlier been one of the driving forces for development of new methods toaccess the vast microbial wealth.

Antimicrobial peptides (AMPs) are evolutionarily conserved and producedby innate immune system in all complex organisms. They serve as thefirst line of defense in humans and mammals. AMPs are polypeptidescomposed of 10-50 amino acids with ≥30% of hydrophobic ratios andpositive charge. In general, AMPs have broadspectrum antimicrobialactivity against bacteria, fungi and yeast. WO 2011/113999 disclosestryptic polypeptides from endophyte of Empetrum nigrum and methods forobtaining them. Based on in silico analysis the peptides are predictedto have antimicrobial activity.

There is an urgent need for new molecules having activity againstvarious pathogenic microbes. This invention meets these needs asexplained in the following.

SUMMARY OF THE INVENTION

Crowberry (Empetrum nigrum) is a perennial shrub growing in the northernhemisphere that has traditionally been used for treating infectiousdiseases and was considered a good candidate for such studies. Thepresent invention provides novel antimicrobial (poly) peptides derivedfrom an endophyte of E. nigrum and variants thereof having improvedactivity against various microbes.

The first aspect of the invention is to provide an antimicrobial peptideor a variant thereof. According to the invention said polypeptidecomprises SEQ ID NO: 5 or has at least 37% identity to SEQ ID NO: 5.

The second aspect of the invention is a method of killing or inhibitinggrowth of microbes. According to the invention said method comprises astep of treating said microbes with a peptide or a variant thereof heredescribed.

The third aspect of the invention is a use of the peptide or a variantthereof here described as a medicament, feed additive, preservative orsurfactant.

Considerable advantages are obtained by the invention. The AMPs areknown to be active against broad spectrum of microbes, including fungiand both Gram positive and Gram negative bacteria. Small AMPs are easyto synthesize and commercially viable as the cost of production isminimal. AMPs are not known to develop resistance in bacteria. Thepresent invention relates to novel antimicrobial peptides, theirvariants and use in various applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. An agar-overlay assay showing subclones with antibacterialactivity (a) and subclones without activity (s) towards S. aureus,identified in the E. coli-based endophytic metagenomic library.

FIG. 2. Predicted amino acid sequence of Empetrum nigrum metagenomicantibacterial protein 1. The predicted protein has 549 residues. Highlyconserved PPR-sequences (pentatricopeptide repeats) are underlined.

FIG. 3. The representative Radial Diffusion assay (RDA) of synthesizedpeptides Met1-Met12, 100 μg each, tested against E. coli (A) and S.aureus (B) NC=negative control, Gentamicin 10 μg.

FIG. 4a shows an alignment of 25 variants of “Met10”-peptide (Chain 100)defined by SEQ ID NO: 4.

FIG. 4b shows an alignment of 23 variants of “Met11”-peptide (Chain 200)defined by SEQ ID NO: 5.

FIG. 4c shows an alignment of 25 variants of “Met12”-peptide (Chain 300)defined by SEQ ID NO: 6.

FIG. 5a shows an alignment of 9 variants of “Met10”-peptide (Chain 100)defined by SEQ ID NO: 4.

FIG. 5b shows an alignment of 10 variants of “Met11”-peptide (Chain 200)defined by SEQ ID NO: 5.

FIG. 5c shows an alignment of 13 variants of “Met12”-peptide (Chain 300)defined by SEQ ID NO: 6.

FIG. 6a . Radial Diffusion assay (RDA) of synthesized peptide variantsChain 100-109, 50 μg each, tested against Escherichia coli (ATCC 25922),Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 10031)and Pseudomonas aeruginosa (ATCC 27853), NC=negative control, Gentamicin5 μg

FIG. 6b . Radial Diffusion assay (RDA) of synthesized peptide variantsChain 200-210, 50 μg each, tested against Escherichia coli (ATCC 25922),Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 10031)and Pseudomonas aeruginosa (ATCC 27853), NC=negative control, Gentamicin5 μg

FIG. 6c . Radial Diffusion assay (RDA) of synthesized peptide variantsChains 300-313, 50 μg each, tested against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC10031) and Pseudomonas aeruginosa (ATCC 27853), NC=negative control,Gentamicin 5 μg

FIG. 7a . Radial Diffusion assay (RDA) of synthesized peptide variantsChains 109, 105, 104, 201, 204, 310, 308, 307 and 306, 20 μg each,tested against Aspergillus flavus (DSM 1959), NC=negative control,Amphotericin B 20 μg.

FIG. 7b . Radial Diffusion assay (RDA) of synthesized peptide variantschains 109, 105, 104, 201, 204, 310, 308, 307 and 306, 20 μg each,tested against Penicillium chrysogenum (DSM 1075), NC=negative control,Amphotericin B 20 μg.

FIG. 8a . Thermostability of the chain peptides 104 (A-D) and 105 (E-H)tested at various temperatures over a period of 14 days by radialdiffusion assay. Antibacterial activity was tested by aliquoting 20 μgof peptides incubated at different temperature and activity was measuredby zone of inhibition. Peptides at −20° C. served as control.

FIG. 8b . Thermostability of the chain peptides 201 (A-D) and 204 (E-H)tested at various temperatures over a period of 14 days by radialdiffusion assay. Antibacterial activity was tested by aliquoting 20 μgof peptides incubated at different temperature and activity was measuredby zone of inhibition. Peptides at −20° C. served as control.

FIG. 8c . Thermostability of the chain peptides 306 (A-D) and 308 (E-H)tested at various temperatures over a period of 14 days by radialdiffusion assay. Antibacterial activity was tested by aliquoting 20 μgof peptides incubated at different temperature and activity was measuredby zone of inhibition. Peptides at −20° C. served as control.

FIG. 9a . pH stability of chain peptides 104 and 105 was determined byradial diffusion assay (RDA). Different pH ranging from 4-9 was used tocheck the zone of inhibition.

FIG. 9b . pH stability of chain peptides 201 and 204 was determined byradial diffusion assay (RDA). Different pH ranging from 4-9 was used tocheck the zone of inhibition.

FIG. 9c . pH stability of chain peptides 306 and 308 was determined byradial diffusion assay (RDA). Different pH ranging from 4-9 was used tocheck the zone of inhibition.

FIG. 10. Narrow spectrum antimicrobial activity of chain peptide 306.

FIG. 11a . Transmission electron microscopy (TEM) image of E. coli (A-C)and S. aureus (D-G) exposed to 10 μg/ml of chain 105 at 37° C. for 1 h.Control (A & D); Treated with chain 105 (B-C & E-G).

FIG. 11b . Transmission electron microscopy (TEM) image of E. coli (A-C)and S. aureus (D-F) exposed to 10 μg/ml of chain 201 at 37° C. for 1 h.Control (A & D); Treated with chain 201 (B-C & E-F).

FIG. 11c . Transmission electron microscopy (TEM) image of E. coli (A-C)and S. aureus (D-F) exposed to 10 μg/ml of chain 308 at 37° C. for 1 h.Control (A & D); Treated with chain 308 (B-C & E-F).

FIG. 12. Antibacterial activity of Chain 105 and 201 immobilizedcatheters. Silicon catheters were used as controls.

DETAILED DESCRIPTION

Endophytes, microorganisms which spend their entire life cycle, or partsof it, inside of healthy tissues of a host plant, are found in all plantspecies. Whereas the majority of endophytes cannot be cultured, it isnot surprising that a chemical structure isolated from a plant canactually have a microbial origin. Metagenomics provides a valuable toolto access the resources of bioactive compounds derived from unculturablemicroorganisms.

In this study, we have constructed a metagenomic library from endophytesand screened for antibacterial activity. The library was screened toselect antimicrobial clones using Staphylococcus aureus as a targetorganism by the double-agar-layer method. One unique clone exhibitingantibacterial activity was selected from the metagenomic library.Secondary libraries were generated to obtain antibacterial subcloneswith reduced insert size for characterization of the gene responsiblefor the antibacterial activity.

The nucleotide sequence of the subclone was subjected to BLAST analysesagainst sequences present in the Genbank databases but no similarity toany known sequence was identified. The isolated gene encoded for aprotein disclosed is in WO 2011/113999 and named as Empetrum nigrummetagenomic antibacterial protein 1 (En-MAP1, GenBank accession numberKC466596). Analysis of the deduced amino acid sequence (SEQ D NO: 1)revealed that En-MAP1 encodes for a protein of 549 amino acids sharingthe highest similarity of 32% to a hypothetical protein from Pseudozymahubeiensis and no similarity to any protein of known function.

The isolated endophytic protein was expressed and isolated but noantimicrobial activity was observed. Antimicrobial activity of the insilico digested peptides was then predicted using algorithms describedin the experimental part. The peptides were also tested forantimicrobial activity against S. aureus, E. coli and Verticilliumdahliae. In preliminary tests some of the peptides predicted to haveantimicrobial activity showed activity also in in vitro tests. Bestcandidates were selected for further studies.

Antimicrobial peptides interact with bacterial membranes to efficientlykill at optimum concentration. AMPs should also possessbiocompatibility, salt tolerance and broad spectrum antimicrobialactivity. By using rational design techniques, we were able to improvethe peptide antimicrobial activity by incorporating or replacing variousamino acids. We have designed and modified Met10 (Chain 100 defined bySEQ ID NO: 4), Met11 (Chain 200 defined by SEQ ID NO: 5), and Met12(Chain 300 defined by SEQ ID NO: 6), peptides with tryptophan (W),arginine (R) and lysine (K) at various positions to improveantibacterial activity.

APD2 database was utilized to check the homology and identities of thepeptides to known antimicrobials (Wang et al., 2009).

An embodiment of the invention is an antimicrobial peptide or a variantthereof comprising SEQ ID NO: 4 (Met 10, chain 100), SEQ ID NO: 5(Met11, chain 200), SEQ ID NO: 6 (Met12, chain 300), or having at least37% identity to SEQ ID NO: SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.It has surprisingly been found that relatively small fragments may havedesired activity.

In one embodiment the peptide or a variant thereof comprises any of SEQID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 or a peptide having at least 40%,preferably at least 50%, 60%, 70% identity to any of said sequences,more preferably at least 80% identity to any of said sequences and mostpreferably 90% or even 95% identity to said SEQ ID NO: 4, SEQ ID NO: 5or SEQ ID NO: 6.

In this connection the term “identity” refers to the global identitybetween two amino acid sequences compared to each other from the firstamino acid to the last corresponding amino acid. Thus a fragment canonly be compared to respective amino acids (essentially equal number ofamino acids) of the mature (poly) peptide.

In one embodiment of the invention the peptide comprising any of SEQ IDNO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 is modified in order to increaseantimicrobial activity.

According to one embodiment the peptide is modified with one or more ofW, R, K, L, C, I, F and A-substitutions, more preferably with one ormore of W, R and A-substitutions. These substitutions increase theinteraction with microbial membranes and thereby improve antimicrobialactivity.

In one embodiment the peptide defined by SEQ ID NO: 4 (chain 100)comprises one or more, preferably, five, six or even seven substitutionsselected from the group consisting of V16K/L/W/R; Q15R/L/W/I/F/C;N14L/K/R/W; /W; A11W; M6W and I7W.

Preferably amino acid residues R8, L9 and H10 of SEQ ID NO: 4 are notmodified.

According to one embodiment the peptide comprises one or moresubstitutions or deletions in SEQ ID NO: 4 (chain 100) selected from thegroup consisting of

-   -   a. D1-/R    -   b. C2-/K    -   c. W3-    -   d. S4-/R    -   e. A5-    -   f. M6W    -   g. I7W    -   h. A11W    -   i. Y13W/R    -   j. N14L/K    -   k. Q15R/L    -   l. V16K/W        wherein “-” indicates deletion of the amino acid.

In one embodiment of the invention the peptide when compared to theoriginal peptide defined by SEQ ID NO: 4 (chain 100), has 1 to 5,preferably 5 N-terminal amino acids been deleted. It has been found thatthe shorter peptides are able to maintain their antimicrobial activity.In addition shorter peptides result in lower cost of synthesis.

In a further embodiment the peptide variant of SEQ ID NO: 4 withN-terminal deletion further comprises one, two, three, four, five, six,seven or all amino acid modifications selected from the group consistingof V16K/W; Q15R/L; N14L/K; Y13R/W; A11W; I7W and M6W.

According to one embodiment the peptide defined by SEQ ID NO: 5 ismodified with one or more of W, R, K, F, I and L-substitutions, morepreferably with one or more of W, K and R-substitutions. Thesesubstitutions increase the interaction with microbial membranes andthereby improve antimicrobial activity.

In one embodiment the peptide defined by SEQ ID NO: 5 (chain 200)comprises one or more, preferably at least six, seven, eight, nine oreven 10 substitutions selected from the group consisting of N1R/K/W,R2W, I3L, V4/I/L, Q5W/K/R, Q6R/L/W, R7W, T8R/W/F, S9F/W/R/L and S10K/L.

Alternatively or in addition the peptide defined by SEQ ID NO: 5 can bemodified by C-terminal addition of K (for example chain 207 defined bySEQ ID NO: 15)

In one embodiment amino acid residue R11 of peptide defined by SEQ IDNO: 5 is not modified.

According to one embodiment the peptide comprises one or moresubstitutions or deletions in SEQ ID NO: 5 (chain 200) selected from thegroup consisting of

-   -   a. N1K/R/W    -   b. R2W    -   c. I3L    -   d. V4I/L    -   e. Q5W/K/R    -   f. Q6R/L/W    -   g. R7W/R    -   h. T8R/W/F/K    -   i. S9F/W/R/L    -   j. S10K/L

According to one embodiment the peptide defined by SEQ ID NO: 6 ismodified with one or more of W, R, K, L, and I-substitutions, morepreferably with one or more of W and R-substitutions. Thesesubstitutions increase the interaction with microbial membranes andthereby improve antimicrobial activity.

In one embodiment the peptide defined by SEQ ID NO: 6 (chain 300)comprises one or more, preferably four, five, six, seven, eight or evennine substitutions selected from the group consisting of Y1I/R/W/K,D2I/L/W/R, G4R/W, F5W/R, G6R/W/L, F8R, K9R, K10R and M11L/R/W/K.

Preferably amino acid residues K3 and L7 of the peptide defined by SEQID NO: 6 are not modified.

Alternatively or in addition the peptide defined by SEQ ID NO: 6 can bemodified by addition of K between amino acid residues 9 and 10 (forexample chain 313 defined by SEQ ID NO: 67).

According to one embodiment the peptide comprises one or moresubstitutions or deletions in SEQ ID NO: 6 (chain 300) selected from thegroup consisting of

-   -   a. Y1I/R/W/K    -   b. D2I/L/W/R    -   d. G4R/W    -   e. F5W/R    -   f. G6R/W/L    -   h. F8R    -   i. K9R    -   j. K10R    -   k. M11L/R/W/K.

In an embodiment the peptide or a variant thereof comprises a sequenceselected from SEQ ID NO: 4 (chain 100) or one of SEQ ID NOs 7 to 31(chains 101 to 125) or consists of it.

In an embodiment the peptide variant (peptide) comprises a sequenceselected from SEQ ID NOs 7 to 15 (chains 101 to 109) or consists of it.In an embodiment the peptide variant comprises a sequence selected fromSEQ ID NOs 10 to 15 (chains 104 to 109) or consists of it. In anembodiment the peptide variant comprises a sequence selected from SEQ IDNOs 10, 11 and 15 (chains 104, 105 and 109) or consists of it.

In an embodiment the peptide comprises SEQ ID NO: 4 (chain 100). In anembodiment the peptide consists of SEQ ID NO: 4 (chain 100).

In an embodiment the peptide or a variant thereof comprises a sequenceselected from SEQ ID NO: 5 (chain 200) or one of SEQ ID NOs 32 to 54(chains 201 to 223) or consists of it.

In an embodiment the peptide variant comprises a sequence selected fromSEQ ID NOs 32 to 41 (chains 201 to 210) or consist of it. In anembodiment the peptide variant comprises a sequence selected from SEQ IDNOs 32, 34, 35 and 39 (chains 201, 203, 204 or 208) or consists of it.

In an embodiment the peptide comprises SEQ ID NO: 5 (chain 200). In anembodiment the peptide consists of SEQ ID NO: 5 (chain 200).

In an embodiment the peptide or a variant thereof comprises a sequenceselected from SEQ ID NO: 6 (chain 300) or one of SEQ ID NOs 55 to 79(chains 301 to 325) or consists of it.

In an embodiment the peptide variant comprises a sequence selected fromSEQ ID NOs 55 to 67 (chains 301 to 313) or consists of it. In anembodiment the peptide variant comprises a sequence selected from SEQ IDNOs 55, 58, 59 and 61 to 65 (chains 301, 304, 305 or 307 to 311) orconsists of it.

In an embodiment the peptide variant comprises a sequence selected fromSEQ ID NOs 61 and 62 (chains 307 and 308) or consists of it.

In an embodiment the peptide comprises SEQ ID NO: 6 (chain 300). In anembodiment the peptide consists of SEQ ID NO: 6 (chain 300).

It is evident that peptides and their variants have differentantimicrobial activities against different microbes. In this studyantimicrobial activity was tested against both bacteria and fungi.

Antibacterial activity of selected peptides (peptide variants) wastested against Escherichia coli (ATCC 25922), Klebsiella pneumoniae(ATCC 10031), Pseudomonas aeruginosa (ATCC 27853) and Staphylococcusaureus (ATCC 25923). Radial diffusion assay and minimum inhibitoryconcentration was used to determine the antibacterial activity, whereradial diffusion assay was used to asses the antifungal activity of thepeptide variants.

Antifungal activity was tested against Aspergillus flavus (DSM 1959) andPenicillium chrysogenum (DSM 1075).

In an embodiment the peptide or a fragment thereof is chemicallysynthesized (synthetic peptide). In another embodiment the peptide hasbeen recombinantly produced. In one embodiment the peptide or peptidesare isolated from the growth medium of recombinant peptide production orsynthesis medium.

In one embodiment, the antimicrobial peptide is attached to a surface ofa medical device, food package, carrier substance or e.g. biosensor,optionally using a linker peptide. Alternatively or in addition, inanother embodiment the peptide is attached to detectable agents such asfor example fluorescent agent or an enzyme substrate for example whenusing the antimicrobial peptide in biosensors. In one embodiment, saidpeptides are immobilized on silicon, polyethene, and other materials inorder to be used as medicine or coating agents.

In hemolysis assays, the ability of peptides at 128 μg/ml concentrationto lyse human erythrocytes to release hemoglobin was measured. Thepeptides described here preferably have low hemolytic activity (0.2 to3.8% hemolysis) in mammal body. The hemolytic concentration tested (128μg/ml) is more than 50 fold of minimal inhibitory concentration (MIC) ofchains 105, 201 and 308 and hemolysis is negligible at 0.2-0.5%respectively. These peptides are useful as antimicrobial drugs as theyshow little or no side effects at concentrations needed to killbacteria.

In one embodiment the peptide or a variant thereof exhibits an activityagainst microorganisms, in particular against gram positive and gramnegative bacteria, in particular against the families Staphylococcaceaeand Enterobacteriaceae. In one embodiment the peptide exhibits anactivity against yeast and/or fungi.

In one embodiment the peptide or a variant thereof exhibits an activityagainst bacteria. In one embodiment the peptide or a variant thereofexhibits an activity against fungi. In one embodiment the peptide or avariant thereof exhibits an activity against bacteria and fungi.Activity against bacteria and/or fungi can be for example suppression ofmultiplication or growth of said microbes or killing them.

In one embodiment the peptide or a variant thereof exhibits an activityagainst gram positive bacteria, in particular microorganisms of thefamily Staphylococcaceae, in particular against microorganisms of thespecies S. aureus.

Peptides comprising essentially any of SEQ ID NOs: 10 to 15 (Chains 104to 109), SEQ ID NOs: 32, 34 to 36, 39 to 41 (Chains 201, 203 to 205, 208to 210) and SEQ ID NOs: 55 to 65 and 67 (Chains 301 to 311 and 313) areparticularly active against Staphylococcaceae, particularly S. aureus.S. aureus has developed resistance to most of the commercially availableantibiotics. Antimicrobial peptides as we describe here, are a promisingclass of new antibiotics, as they do not induce resistance inmicroorganisms.

In one embodiment the peptide exhibits an activity against gram negativebacteria, in particular against E. coli. Peptides comprising essentiallyany of SEQ ID NOs: 10 to 15 (Chains 104 to 109), SEQ ID NOs: 32, 34 to36, 39 to 41 (Chains 201, 203 to 206, 208 to 210) and SEQ ID NOs: 55 to65 and 67 (Chains 301 to 311 and 313) are particularly active against E.coli.

In one embodiment the peptide exhibits an activity against Klebsiella,particularly Klebsiella pneumoniae. Peptides comprising essentially anyof SEQ ID NOs: 10 to 15 (Chains 104 to 109), SEQ ID NOs: 32, 34, 35, 39to 41 (Chains 201, 203, 204, 208 to 210) and SEQ ID NOs: 55, 58, 59, 61to 65, and 67 (Chains 301, 304, 305, 307 to 311 to 311 and 313) areparticularly active against Klebsiella.

In one embodiment the peptide exhibits an activity against Pseudomonas,particularly Pseudomonas aeruginosa. Peptides comprising essentially anyof SEQ ID NOs: 10 to 12 and 15 (Chains 104 to 106, 109), SEQ ID NOs: 32,34, 35 and 39 (Chains 201, 203, 204, 208) and SEQ ID NOs: 55 to 65 and67 (Chains 301 to 311 and 313), are particularly active againstPseudomonas.

In one embodiment the peptide exhibits an activity against Aspergilli,particularly A. flavus. Peptides comprising essentially any of SEQ IDNO: 10, 11 or 15, SEQ ID NO: 32 or 35 (Chains 104, 105 or 109), SEQ IDNOs: 32 or 35 (chains 201 or 204); SEQ ID NOs: 60, 61, 62 and 64 (chains306, 307, 308 and 310) are particularly active against Aspergilli.

In one embodiment the peptide exhibits an activity against Penicillium,particularly P. chrysogenum. Peptides comprising essentially any of SEQID NO: 10, 11 or 15, SEQ ID NO: 32 or 35 (Chains 104, 105 or 109), SEQID NOs: 32 or 35 (chains 201 or 204); SEQ ID NOs: 60, 61, 62 and 64(chains 306, 307, 308 and 310) are particularly active againstPenicillium.

In one embodiment the peptide exhibits an activity against clinicalstrains of E. coli, K. pneumoniae, E. cloacae, P. aeruginosa, Serratiamarcescens, Proteus mirabilis, S. pneumoniae, Acinetobacter baumannii,A. johnsonii, S. aureus, Candida albicans, C. glabrata, C. parapsilosisand C. quillermondiae. Peptides comprising essentially any of SEQ ID NO:10, 11 or 15, SEQ ID NO: 32 or 35 (Chains 104, 105 or 109), SEQ ID NOs:32 or 35 (chains 201 or 204); SEQ ID NOs: 58, 60, 61, 62, 63, 64 and 65(chains 304, 306, 307, 308, 309, 310 and 311).

Chain 104, chain 105, chain 109, chain 201, chain 204, chain 307, chain308 and chain 310 are demonstrably broad-spectrum antimicrobialpeptides, active against gram positive and negative bacteria in a rangeof 0.5 to 32 μg/ml and has little side effects to humans as tested byhemolysis assay.

The present invention relates also to an antimicrobial compositioncomprising an antimicrobial peptide or peptides here described. In anembodiment such composition further comprises a pharmaceuticallyacceptable carrier and optionally other conventional ingredients.

Peptide having SEQ ID NO: 4 (chain 100), SEQ ID NO: 5 (chain 200), SEQID NO: 6 (chain 300) or a variant thereof having at least 37%,preferably at least 40%, 50%, 60%, 70%, more preferably at least 80% andmost preferably at least 90% identity to said sequence or any peptideaccording to the present disclosure for use in therapy, particularly foruse as an antimicrobial agent.

The antimicrobial activity can be against bacteria or fungi or bacteriaand fungi. The bacteria can be gram positive or gram negative bacteria.In particularly the peptide has activity against microorganisms of thegenera Staphylococcus or E. coli or both Staphylococcus and E. coli.Various antimicrobial activities and hemolytic properties of thepeptides have been discussed above.

One further embodiment of the invention is a use of the peptideaccording to the present disclosure as a medicament, feed additive,preservative or surfactant.

In one embodiment a peptide having any of SEQ ID NO: 4, 5 or 6 or avariant thereof having at least 37%, preferably at least 40%, 50%, 60%,70%, more preferably at least 80% and most preferably at least 90%identity to said sequence or any peptide according to the presentdisclosure is used as a medicament, especially as a medicament againstcontaminating microbes including S. aureus discussed above.

The present invention relates also to a use of the peptide(s) and theirvariants described here as a medicament, feed additive, preservative orsurfactant is claimed. The toxins produced by various microorganismscontaminate human foods and animal feeds and these peptides used aspreservatives may reduce the growth of the contaminants. The activitiesand other properties of the peptides have been discussed above inconnection of other embodiments.

Peptides chain 104, chain 105, chain 109, chain 201, chain 204, chain307, chain 308 and chain 310 are demonstrably broad-spectrumantimicrobial peptides, active against gram positive and negativebacteria in a range of 0.5 to 32 μg/ml and has little side effects tohumans as tested by hemolysis assay. These peptides has a potential fortreatment against gram negative infections caused by Escherichia coli(ATCC 25922), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa(ATCC 27853) and gram positive Staphylococcus aureus (ATCC 25923).

The present invention relates also to a method of killing or inhibitinggrowth of microbes, comprising the step of treating said microbes with apeptide(s) or their variants described here. In one embodiment thepeptide or a variant there of exhibits an activity againstmicroorganisms, in particular against gram positive and/or gram negativebacteria, in particular against the families Staphylococcaceae andEnterobacteriaceae, in particular against microorganisms of the speciesS. aureus and/or E. coli. In one embodiment the peptide or a variantthereof exhibits an activity against fungi. In still further embodimentthe peptide or a variant thereof exhibits activity against both fungiand bacteria. The activities and other properties of the peptides havebeen discussed above in connection of other embodiments.

Catheter acts as a carrier of bacteria through attachment to the surfaceand then to the lumen of urethra. Most of the catheters are made ofsilicone rubber where microbes adhere easily to form biofilms and causeinfections. Several strategies have been developed to overcome theseproblems by using antibiotic compounds such as rifampin and minocycline,or silver coating on catheter surfaces. However, the use of antibioticsrisk development of bacterial resistance and antibiotic efficacy in theclinical applications is not well known. Silver-coated catheters werefound ineffective in in-vitro studies.

Catheter-associated urinary tract infections (CAUTI) can be prevented byusing antimicrobial peptide (AMP)-coated urinary catheters, which alsoprevent the spread of antibiotic resistance.

It should also be understood that the peptide(s) or their variants ofthe present invention can be used with various uses requiringantimicrobial activity.

It is to be understood that the terminology employed herein is for thepurpose of description and should not be regarded as limiting.

The features of the invention described here as separate embodiments mayalso be provided in combination in a single embodiment. Also variousfeatures of the invention described here in the context of a singleembodiment, may also be provided separately or in any suitablesub-combination. It should be understood, that the embodiments given inthe description above are for illustrative purposes only, and thatvarious changes and modifications are possible within the scope of thedisclosure.

A listing of the cited references will be given below. The contents ofall citations are herewith incorporated by reference.

The following non-limiting examples illustrate the invention.

EXAMPLES Example 1: Total Plant DNA Isolation

Fresh and young leaves of Empetrum nigrum L. were surface sterilizedwith 70% ethanol (v/v) for 1 min and in sodium hypochlorite (3.5% v/v)for 5 min and used for the isolation of genomic DNA (Pirttilä et al.2001).

Example 2: Metagenomic Library Construction and Screening forAntibacterial Activity

Plant DNA isolated from E. nigrum was dissolved in sterile water to aconcentration of 0.1 μg μl⁻¹. The DNA was then subjected to preparativepulsed-field gel electrophoresis in a CHEF-DRII (Bio-Rad) system. Theelectrophoresis conditions (pulse intervals and durations) were: N/S—60s and E/W—60 s for 6 h; N/S—90 s and E/W—90 s for 6 h; N/S—99 s andE/W—99 s for 6 h, respectively, with a voltage of 6 V/cm, a 120° fixedangle and using a 0.15× Tris-borate-EDTA (TBE) buffer. During theelectrophoresis, the temperature was maintained at 10° C. Afterelectrophoresis, a strip from each side of the gel was cut off andstained with ethidium bromide to visualize the DNA. Thehigh-molecular-weight DNA was then excised from the remaining unstainedpart of the gel and electro-eluted for 1 h at 100 V in a dialysis bagcontaining 0.5×TBE. Amplification by primers specific for fungi andbacteria was done as previously (Koskimäki et al. 2010, Tejesvi et al.2010) to confirm the separation of microbial DNA. For cloning, the DNAof about 25-30 kb was end repaired to produce 5′-phosphorylated DNA andligated to blunt-ended dephosphorylated pCC1FOS™ vector. The ligationmixture was packaged into lambda phages using MaxPlax Lambda PackagingExtracts (Epicentre). The packaged library was then transduced into E.coli EPI-300, and the transformants were selected on LB agar platessupplemented with chloramphenicol. The packaged fosmid library wasstored in cryotubes as clone pools containing approximately 10³ clonesper pool until screening.

The fosmid library screening was performed as follows: clone pools werethawed and spread onto 150-mm LB agar plates supplemented withchloramphenicol to obtain ˜1000 colonies per plate. The library plateswere incubated overnight at 30° C. followed by incubation at roomtemperature (RT) for additional 3-5 days. The plates were overlaid withtop agar containing exponentially growing Staphylococcus aureus andincubated overnight at 37° C., followed by further incubation at RT for3-5 days. Colonies with antibacterial activity were identified by a zoneof inhibition of S. aureus growth. Such colonies were picked through thetop agar and separated from the chloramphenicol-sensitive assay strain(S. aureus) by streaking onto LB plates containing ampicillin andchloramphenicol. Restriction analysis of the selected antibacterialfosmid clone was carried out by digestion with BamHI andelectrophoresis.

Example 3: Strains, Plasmids and Growth Conditions

EPI-300™-T1® Phage T1-resistant E. coli cultures were grown at 37° C. onLuria-Bertani (LB) agar or in LB broth+10 mM MgSO₄ supplemented with theappropriate antibiotics. The following antibiotic concentrations wereused for the E. coli strain: chloramphenicol 12.5 μg ml⁻¹ and ampicillin100 μg ml⁻¹. Plasmid pCC1FOS™ (Epicentre, Madison, USA) that carries twoorigins of replication, a single copy origin (ori2) and an induciblehigh copy origin (oriV) was used to construct the metagenomic libraryfrom endophytes of Empetrum nigrum and for subcloning the genesconferring such antibacterial activity. The pET11-c vector was used toexpress the genes responsible for the antibacterial activity in the hoststrain E. coli BL21 (DE3) gold.

Example 4: Subcloning and Sequencing of Clone pFosS1A

The antibacterial fosmid clone selected from the agar overlay assay wasnamed pFosS1. The metagenomic fosmid was isolated using the PlasmidMidiprep Kit (Qiagen) and subjected to partial digestion with Sau3AI(0.1 U μl⁻¹ of DNA, 37° C. for 15 min) and electrophoresis for sizeselection of the DNA and subcloning. Fragments greater than 1.5 kb wereextracted from the gel, end-repaired and ligated into blunt-endeddephosphorylated pCC1FOS™. The ligation mixture was transformed into E.coli and the recombinant clones were screened by the agar overlay assayand spread onto LB plates supplemented with chloramphenicol. Thesubclones showing clear zones of inhibition of S. aureus were analysedby gel electrophoresis after digestion with BamHI to select the subcloneharboring the smallest insert of ˜1.8 kb. This subclone was namedpFosS1A and sequenced with the primers pCC1 forward and pCC1 reverseaccording to the manufacturer's instructions (Abi 3730 DNA Analyser, AbiPrism BigDye Terminator Cycle Sequencing Kit, Applied Biosystems,Warrington, UK). The open reading frame contained within the subclonepFosS1A was named En-MAP1 and analyzed by the BLAST program of Genbankas well as SignalP and pfam.

Example 5. Construction and Screening of the Metagenomic Library

The band corresponding to endophytic DNA was separated from the Empetrumnigrum genomic DNA, which remained in the wells of the agarose gel afterPFGE electrophoresis. Presence of endophytic DNA in the band wasconfirmed by amplification of PCR products with primers specific forfungi and bacteria. Approximately 8,000 metagenomic clones were obtainedfrom 20 μg DNA. Screening of the metagenomic library by the agar overlaymethod resulted in the identification of one antibacterial cloneexhibiting an inhibition zone for S. aureus. However, growth inhibitionof the host E. coli was not observed. Restriction fragment analysisrevealed that the clone carried an insert DNA of over 30 kb in size. Asecondary library was generated from the antibacterial clone to selectantibacterial subclones and to characterize the individual gene(s)responsible for the antibacterial activity. Restriction fragmentanalysis of antibacterial subclones identified the smallest insert of1.8 kb in subclone pFosS1A, which still exhibited growth inhibition ofS. aureus in the agar overlay assay (FIG. 1).

Example 6: Fractionation and Analysis of Clone Supernatant

E. coli cells carrying the subclone pFosS1A and control (empty vector)were grown overnight at 37° C. in LB-broth containing chloramphenicol.These cultures were used as inocula for the copy number amplificationprocedure. One volume of these cultures was added to 10 volumes of freshLB+chloramphenicol and 1/100 of CopyControl Induction Solution(Epicentre) was added to the media to induce clones to high copy number.After vigorous shaking of cultures at 37° C. for 20 h, the supernatantwas separated from bacterial cells by centrifugation for 20 min at3040×g at 4° C. The supernatant was freeze-dried by Heto PowerDry LL1500freeze dryer (ThermoElectron, Mukarov, Czech Republic). The freeze-driedbroth was weighed and 50 mg was dissolved to 2 ml of distilled water.The material was kept in ultrasonicator for 20 minutes, centrifuged for10 minutes, filtered (GHP Bulk Acrodisc 13, Pall Life Sciences) andfractioned by semi preparative High Pressure Liquid Chromatography(HPLC). The fractions were then tested for antibacterial activity by the96-well plate standard method using S. aureus as the test strain.Supernatants of subclone pFosS1A and control were analyzed for smallmolecules by Alliance 2690 HPLC (Waters, Milford, Mass., USA) combinedwith Micromass LCT Time-of-flight mass spectrometry (TOFMS) (Micromass,Altrincham, UK).

Example 7. Sequence Analysis of the Antibacterial Subclone

The insert of subclone pFosS1A was completely sequenced in bothdirections and it contained a unique open reading frame of ˜1650 bp. Theputative ribosome-binding site and promoter sequence are present at˜35/−10 upstream region from the initiation codon. The nucleotidesequence was subjected to BLAST analyses against sequences present inthe Genbank databases but no similarity to any known sequence wasidentified. This suggested that the isolated gene encoded for a proteinof novel structure and therefore the protein was named Empetrum nigrummetagenomic antibacterial protein 1 (En-MAP1). Analysis of the deducedamino acid sequence revealed that En-MAP1 encodes for a protein of 549amino acids (FIG. 2), sharing the highest similarity of 32% to ahypothetical protein from Pseudozyma hubeiensis and no similarity to anyprotein of known function. SignalP analysis indicated that the aminoacid sequence of En-MAP1 had no putative amino-terminal signal sequencesfor secretion or translocation. When the deduced amino acid sequence wasanalyzed for conserved motifs using the pfam protein family database,three pentatricopeptide repeat-motifs were identified at positions 77 to107, 126 to 153 and 392 to 420 aa (FIG. 2). En-MAP1 was predicted to bea soluble protein having no transmembrane region analyzed by TMpred andPSORT. The residues 147-176 also consisted a predicted coil-coil region,which is typical for proteins involved in protein-protein interactions,and a possible leucine zipper starting at residue 490, analyzed byPSORT.

Example 8: Protein Expression in pET23(b)

The gene En-MAP1 was amplified using primers with Nde1 and Sal1restriction sites pFosS1F (CATATGAGACTAGTAGCTCATCCTGTTCCTGATGC; SEQ IDNO: 2) and pFosS1R (GTCGACTTATTAACGAGATGACGTCCTCTGCTGTACG; SEQ ID NO:3). The amplification products were cloned into pET23(b) vectors,transformed into XL1 competent cells, and the gene identity wasconfirmed by sequencing. The expression studies were done using E. colistrains BL21 pLysS and BL21 pRARE as the hosts. The protein wasexpressed in both strains as inclusion bodies, which were isolated (vanLith et al. 2007). The inclusion bodies were suspended in 5M guanidinehydrochloride/0.2 M sodium phosphate buffer (pH 7.0) and the protein wasrefolded on HisTrap Column (GE Healthcare Life Sciences) using a linearbuffer exchange from 3 M guanidine/0.2 M sodium phosphate (pH 7.0) to0.2 M sodium phosphate (pH 7.0) over 4 h in AKTA FPLC, and finallyeluted with 50 mM EDTA/20 mM sodium phosphate buffer (pH 7.0). Thenucleotide sequence of En-MAP1 has been deposited to the GenBank underaccession number KC466596.

Example 9: Antibacterial Activity Analysis of Protein

Both folded and unfolded proteins were tested for antibacterial activityagainst S. aureus by pipetting 10 μg of protein on the S. aureus cultureon an LB plate. The activity was also tested in folded and unfoldedprotein digested with trypsin in 0.01% acetic acid at concentrations of30 and 60 μg/ml.

Example 10: Prediction of Antimicrobial Peptides and Peptide Synthesis

The protein En-MAP1 was in silico-trypsin digested<(http://au.expasy.org)> and 12 peptides (Met1-Met12) were predicted<(http://www.bicnirrh.res.in/antimicrobial)> to be antimicrobial bythree different algorithms (Support Vector Machine (SVM) classifier,Random Forest Classifier and Discriminant Analysis Classifier) and byAPD2 <(http://aps.unmc.edu/AP/prediction/prediction_main.php)>.Synthesized peptides were purchased from GenScript, USA.

Example 11: Antimicrobial Activity of Synthesized Peptides

The radial diffusion assay (RDA) was carried out as described byAndersen et al. (2010). Briefly, 30 ml of 1/10 Muller-Hinton broth (MHB)supplemented with 1% agarose and 5.0×10⁵ CFU/ml S. aureus or E. coli orK. pneumoniae, P. aeruginosa cells was poured into a single-wellomnitray (Nunc) and overlaid with a TSP 96-well plate. One hundred μg ofeach synthesized peptide was tested. The peptides were also testedagainst Fusarium oxysporum and Verticillium dahliae, for which ⅓ potatodextrose agarose was used. To test the synergistic effect of peptides,each peptide was mixed in equimolar concentrations. Mix1 (met1-12), Mix2(met3, 4 and 5), Mix3 (met 8, 9 and 10), Mix 4 (met11, 12) and Mix5 (8,9, 10, 11, 12) were prepared and tested against E. coli and S. aureus.Gentamicin and vancomycin were included as positive controls forbacteria and amphotericin B was used as the control for fungi.

Minimum inhibition concentrations (MICs) were determined againstEscherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923),Klebsiella pneumoniae (ATCC 10031) and Pseudomonas aeruginosa (ATCC27853). Also clinical strains of E. coli, K. pneumoniae, E. cloacae, P.aeruginosa, Serratia marcescens, Proteus mirabilis, S. pneumoniae,Acinetobacter baumannii, A. johnsonii and S. aureus were used for MICsassays. Additionally, clinical strains of yeast including Candidaalbicans, C. glabrata, C. parapsilosis and C. quillermondiae were used.The susceptibility of these microorganisms to Chain peptides weredetermined using micro broth dilution assay. Colonies of the respectivemicroorganism from Muller Hinton (MH) agar plates incubated overnightwere suspended in MH broth media and final concentration ofmicroorganism was adjusted to 5.0×10⁵ cfu/ml. An aliquot of 10 μl ofeach Chain peptide, or gentamicin, tetracycline as reference antibioticswere added in differing concentrations along with 90 μl of bacterialsuspension to 96 well polypropylene plates and incubated at 37° C. for20 to 24 hours. Concentrations of Chain peptides and referenceantibiotics ranged from 0.125 to 128 μg/ml and the MICs were recorded asthe lowest concentration with no visual growth of bacteria. Similarly,antifungal MICs were tested in Potato dextrose broth.

Example 12: Expression Analysis of the Antibacterial Clone

The subclone expressing pFosS1A was analyzed by HPLC-MS for possibleproduction of small antimicrobial molecules in the liquid medium andcompared with the control (empty vector). The chromatograms and spectraof the peaks for the antibacterial clone and the control were identical.The medium was fractioned and tested for antibacterial activity againstS. aureus, but no activity was detected. This suggested that theantibacterial activity against S. aureus was not from a metabolitegenerated by En-MAP1 activity.

The gene En-MAP1 was then cloned into the expression plasmid pET23(b) toanalyze whether the antibacterial activity was due to the proteinitself. The resulting construct pET23(b)-FosS1A contained the En-MAP1gene in-frame. When transformed into E. coli expression strain BL21(DE3) pLyseS and BL21 (DE3) pRARE, a protein of approximately 63 kDa(predicted mass 63.3 kDa) was produced in the cell extracts of induced,but not in the uninduced cultures. The resulting protein includes theamino acid sequence MHHHHHHM- (SEQ ID NO: 80) prior to the first aminoacid of the protein sequence. The protein was expressed in both strainsas inclusion bodies and they were purified and folded in a His-Trapcolumn, resulting in about ˜1 mg of pure folded protein. The foldedprotein was tested for antibacterial activity against S. aureus and noactivity was observed. To test whether a shorter fragment wasresponsible for the antibacterial activity due to alternativetranslation starting sites, three constructs encoding shorter fragmentsof En-MAP1 were designed and expressed, but no activity was observed.However, when the folded full En-MAP1 protein (SEQ ID NO: 1) wasdigested with trypsin, antibacterial activity was observed for up to twohours after digestion (data not shown). No activity was detected in thetrypsin digest of the unfolded protein. This suggested that theantimicrobial activity resulted from fragmentation of the protein,producing a peptide, which contained an internal trypsin cleavage sitethat was protected by the folded structure.

To further identify the peptide responsible for antimicrobial activity,the En-MAP1 protein was digested in silico and the peptides, which werepredicted to be antimicrobial, were synthesized. Cleavage sites forseveral restriction enzymes, proteases and chemical compounds, such asArgC proteinase, Asp-N endopeptidase (EC 3.4.24.33), chymotrypsin-HS30(EC 3.4.21.1), clostripain, CNBr (EC 208-051-2), formic acid, Lys-C,iodosobenzoic acid, proline endopeptidase, and trypsin were included inthe in-silico analysis. Antimicrobial activities of the peptidefragments were predicted using four different algorithms (Support VectorMachine (SVM) classifier, Random Forest Classifier, DiscriminantAnalysis Classifier and APD2).

The majority of these peptides were from tryptic digest predictions,while three peptides (shown as Met10, Met11 and Met12 in FIG. 3 andChain 100 defined by SEQ ID NO: 4, Chain 200 defined by SEQ ID NO: 5 andChain 300 defined by SEQ ID NO: 6, respectively in tables (1, 2a, 2b,2c, 3a, 3b and 3c) and sequence listing) resulted fromAsp-N-endopeptidase16, chymotrypsin-HS30 and CnBr digestions,respectively. Eight of these peptides contained an internal trypsincleavage site.

Example 13: Salt, pH and Thermal Stability

Thermal stability was evaluated by diluting peptides to theconcentration of 2 mg/ml in PBS buffer (pH 7.4) and incubating atdifferent temperatures, such as +4° C., 25° C. (RT), 37° C. and 45° C.,peptides stored at −20° C. were used as control. After each timeinterval, 100 μl (2 mg/ml) of peptides were taken and stored at −20° C.for radial diffusion assay to determine the antibacterial activity (FIG.8). Peptides were also tested by diluting them at different pH valuesstarting from pH 4 to 9. The effect of NaCl on the antimicrobialactivity was tested by adding different concentrations (50, 100 and 200mM) of NaCl to MH medium and the minimum inhibitory concentrations weretested against 4 ATCC bacterial strains.

Example 15: Bacterial Adherence on Coated and Uncoated Silicon Catheters

The coated (Chain 105 or Chain 201) and uncoated silicon catheters werecut into 0.5 cm pieces and placed in 24 well plates suspended with 1 mlof 5×10⁵ CFU/mL bacterial (E. coli and S. aureus) culture in phosphatebuffer saline (PBS). Samples were incubated at 37° C. for 6 h at 150RPM. After incubation, each catheter piece was rinsed with 1 ml of PBSfor 2 times using fresh PBS. The catheter pieces were transferred toEppendorf tubes and 500 μl of PBS was added, sonicated in water bath for2 minutes, vortexed for 5 sec, serially diluted, and CFUs weredetermined.

Example 14: Preparation of Cells for Transmission Electron Microscopy(TEM)

E. coli and S. aureus were grown to mid-exponential phase and diluted inMuller Hinton broth (MHB) medium to a cell density of 0.1 by takingabsorbance at 600 nm and incubated at 37° C. for 1 h with 10 μg/ml Chainpeptides. After incubation, an equal volume of 2% glutaraldehyde in 0.1M phosphate buffer was added and the cells were pelleted bycentrifugation at 5000 rpm for 2 minutes, and the cell pellet was fixedwith 1% glutaraldehyde in 0.1 M phosphate. Cells were post-fixed withosmium tetroxide, dehydrated using increasing concentrations of acetoneor alcohol, and embedded in plastic resin (Epon). Ultrathin sections(70-80 nm) were post-stained with uranyl acetate and lead citrate beforeobservation with TEM. Microscopy was performed with a Tecnai G2 Spirit120 kV TEM with Veleta and Quemesa CCD cameras.

TABLE 1 The peptide variants generated using tryptophan, lysine andarginine residues at various places and predicted by statistical modelsusing CAMP database (SVM, RF, ANN and DA). Ali- Sl. SVM RF DA phaticInstability Net CAMP No Name Sequence values^(a) values^(b) ANN^(c)values^(d) Index^(e) Index^(f) charge Identities database^(g) 4 Chain100DCWSAMIRLHAKYNQV 0.156 0.4615 AMP 0.012 79 14.7 1 6/11 (54%) CAMPSQ40687 Chain101 KIRLHRKRLRK 0.976 0.725 AMP 0.995 106 48 7 6/11 (54%)CAMPSQ73 8 Chain102 KKRLHRKRLRK 0.997 0.6695 AMP 0.995 70 85 8 8/11(72%) gi_33736048 9 Chain103 KLRLHAKRLRK 0.893 0.696 AMP 0.922 115 66 6No hits nil 10 Chain104 RKWRAMIRLHAKRLRK 0.989 0.7955 AMP 0.871 85 41 86/11 (54%) gi_74472293 11 Chain105 RKWRAMIRLHAKWLRK 0.997 0.8375 AMP0.852 85 29 7 6/11 (54%) gi_67584689 12 Chain106 WIRLHWKRLRK 1 0.927 AMP0.995 106 53 5 5/11 (45%) gi_59754113 13 Chain107 WWRLHAKKKLW 1 0.97 AMP0.993 80 22.9 4 4/11 (36%) CAMPSQ4044 14 Chain108 WWRLHAKRKLW 1 0.936AMP 0.994 80 52 4 6/11 (54%) CAMPSQ4044 15 Chain109 WWRLHAKWKLW 1 0.9765AMP 0.996 80 22 3 4/11 (36%) gi_59754171 16 Chain110 KLKRAMIRLHAKKRLK0.916 0.8285 AMP 0.926 110 34 8 7/16 (44%) gi_75999248 17 Chain111KLKRAMIRLHAKKWRW 0.996 0.869 AMP 0.816 85 54 7 No hits nil 18 Chain112RLKRAMIRLHAKKWRW 0.995 0.861 AMP 0.826 85 60 7 No hits nil 19 Chain113RWWRAMIRLHAKKWRW 1 0.8495 AMP 0.97 61 80 6 7/16 (44%) gi_59754035 20Chain114 WWRLHAAKKIL 1 0.9625 AMP 0.991 124 19 3 5/11 (45%) CAMPSQ413721 Chain115 WWRLHAKKKCW 0.998 0.9205 AMP 0.97 45 30 4 4/11 (36%)CAMPSQ4044 22 Chain116 WWRLHAKKKFW 1 0.954 AMP 0.995 45 9.09 4 4/11(36%) CAMPSQ4044 23 Chain117 WWRLHAKKKIW 1 0.969 AMP 0.993 80 1.37 43/11 (27%) CAMPSQ4009 24 Chain118 WWRLHAKKKRW 1 0.9075 AMP 0.996 45 90 55/11 (45%) gi_3407608 25 Chain119 WWRLHAKKKWR 1 0.907 AMP 0.993 45 9 54/11 (35%) gi_59754211 26 Chain120 WWRLHAKKKWW 1 0.968 AMP 0.998 45 9.094 5/11 (45%) CAMPSQ4044 27 Chain121 WWRLHAKLKLW 1 0.9585 AMP 0.995 1157.4 3 4/11 (36%) CAMPSQ4044 28 Chain122 WWRLHAKRKRW 1 0.8155 AMP 0.99744 120 5 5/11 (45%) gi_3407608 29 Chain123 WWRLHAKWRWR 1 0.928 AMP 0.99844 61 4 6/11 (54%) gi_59754169 30 Chain124 WWRLHARKRWW 1 0.933 AMP 0.99944 90 4 5/11 (45%) gi_3407608 31 Chain125 WWRLHAWKWRR 1 0.936 AMP 0.99844 61 4 7/11 (63%) gi_59754006 5 Chain200 NRIVQQRTSSR 0.028 0.3175 NAMP0.006 61 53 3 No hits found nil 32 Chain201 KWIVWRWRFKR 1 0.977 AMP 0.9661 69 5 6/11 (54%) gi_59754085 33 Chain202 RKIVKKRTFKR 0.988 0.6975 AMP0.997 61 47 7 6/11 (54%) gi_33735965 34 Chain203 RRIVKLRWFKR 1 0.8035AMP 0.944 97 128 6 No hits nil 35 Chain204 RRLIWRRFKWLR 1 0.889 AMP0.978 97 118 6 5/11 (45%) gi_59754033 36 Chain205 KRIVRWRTRKR 0.995 0.72AMP 0.956 61 112 7 4/11 (36%) gi_3407606 37 Chain206 KRIVRWRWRKR 1 0.787AMP 0.912 61 164 7 5/11 (45%) gi_59754033 38 Chain207 KRIVRWRKLKRK 0.9990.8415 AMP 0.943 89 90 8 6/11 (54%) CAMPSQ3545 39 Chain208 WRILRWRKLKR 10.9165 AMP 0.984 106 110 6 5/11 (45%) CAMPSQ3545 40 Chain209 WRIVRWRKLKR1 0.9055 AMP 0.971 97 67 6 6/11 (54%) CAMPSQ3545 41 Chain210 WRIVQWRKLKR0.999 0.896 AMP 0.844 97 15 5 4/11 (36%) gi_59754101 42 Chain211KRIVRRRTFKR 1 0.6455 AMP 0.999 61 162.35 7 6/11 (54%) gi_75999237 43Chain212 KRWRKWRLFKR 1 0.803 AMP 0.95 35 106 7 5/11 (45%) gi_74472319 44Chain213 NRIVLLRTFKR 0.752 0.6575 AMP 0.983 132 28 4 No hits nil 45Chain214 NRIVKKRTFKR 0.937 0.6525 AMP 0.983 132 28 4 6/11 (54%)CAMPSQ3535 46 Chain215 RKIVKRRTFKR 0.997 0.672 AMP 0.998 61 99 7 No hitsnil 47 Chain216 RKIVWWRTFKR 0.997 0.8025 AMP 0.963 61 20 5 4/11 (35%)gi_62788246 48 Chain217 RLIVRRRTFKR 0.999 0.678 AMP 0.998 97 132 6 Nohits nil 49 Chain218 RRIVRKKTFKR 0.997 0.652 AMP 0.968 61 80 7 6/11(54%) gi_75999276 50 Chain219 RRIVWRRTFKR 1 0.6675 AMP 0.977 61 132 64/11 (36%) CAMPSQ3545 51 Chain220 RWIVQRRTFKR 1 0.679 AMP 0.986 61 184 6No hits nil 52 Chain221 RVIVRRRTFKR 0.999 0.6545 AMP 0.996 88 132 6 Nohits nil 53 Chain222 WKIVKKRTRRR 0.991 0.788 AMP 0.985 61 124 7 4/11(36%) gi_74472305 54 Chain223 WRIVRRRTFKR 0.999 0.6965 AMP 0.989 62 1326 4/11 (36%) gi_3407608 6 Chain300 YDKGFGLFKKM 0.705 0.2515 AMP 0.11 3531 2 No hits nil 55 Chain301 IIKRFRLFKKL 0.989 0.9255 AMP 0.999 141 10 55/11 (45%) gi_115794206 56 Chain302 ILKRWWLFKKL 1 0.9715 AMP 0.996 14191 4 5/11 (45%) gi_59754119 57 Chain303 IWKRFRLFKKR 1 0.883 AMP 0.976 7155 6 5/11 (45%) gi_115794207 58 Chain304 IWKRFRLFKKW 1 0.955 AMP 0.98 7125 5 5/11 (45%) CAMPSQ754 59 Chain305 RLKWFWLRKLK 0.999 0.95 AMP 0.947106 14.7 5 4/11 (36%) gi_59754121 60 Chain306 RLKRWRLFRKR 1 0.6875 AMP0.917 70 134 7 6/11 (54%) gi_62788246 61 Chain307 RLKWFWLFRKR 0.9990.8795 AMP 0.946 106 60.7 6 5/11 (45%) gi_59754067 62 Chain308RLKWFLLFRKR 0.992 0.8335 AMP 0.94 106 31 5 5/11 (45%) gi_27291218 63Chain309 WRKWFWLFKKR 1 0.9455 AMP 0.995 35.4 36.2 5 6/11 (54%) CAMPSQ35464 Chain310 KRKWRWLFKKL 0.999 0.8745 AMP 0.944 70 28 5 6/11 (54%)CAMPSQ461 65 Chain311 KLKWFWLFKKR 0.999 0.954 AMP 0.91 70 21 5 4/11(36%) gi_10064836 66 Chain312 KLKKFKLFKKR 0.999 0.819 AMP 0.96 70 −11.57 8/11 (72%) gi_115794212 67 Chain313 RLKRFRLFRKRK 0.999 0.722 AMP 0.99465 56 8 5/12 (42%) gi_115794207 68 Chain314 KRKRFRLFKKR 1 0.6525 AMP0.998 35 84 8 6/11 (54%) gi_115794207 69 Chain315 RLKRFRLFKKL 0.9970.7565 AMP 0.982 106 9.6 6 5/11 (45%) gi_115794207 70 Chain316RRKRFRLFKKM 0.985 0.5865 AMP 0.998 35 106 7 7/11 (63%) CAMPSQ114 71Chain317 RRKRFRLFRRK 1 0.639 AMP 0.998 36 142 8 7/11 (63%) gi_11206272072 Chain318 RWKRFRLFKKR 1 0.7545 AMP 0.952 35 106 7 5/11 (45%)gi_115794208 73 Chain319 RWKRFRLFKKW 1 0.8795 AMP 0.954 35 77 6 5/11(45%) gi_115794208 74 Chain320 WKKGFGLFKKM 0.998 0.713 AMP 0.967 35 17 46/11 (54%) CAMPSQ3086 75 Chain321 WKKRFRLFKKL 1 0.878 AMP 0.905 70 17 66/11 (54%) CAMPSQ3086 76 Chain322 WLRRFRLFRRL 1 0.769 AMP 0.986 106 1415 6/11 (54%) gi_62788246 77 Chain323 RLKRFLLFRKRL 0.996 0.687 AMP 0.988130 56 6 7/12 (58%) CAMPSQ2349 78 Chain324 KRKWFWLFKKL 0.999 0.8745 AMP0.944 70 28 5 6/11 (54%) CAMPSQ461 79 Chain325 KLKRFRLFKKR 0.998 0.712AMP 0.977 70 39 7 6/11 (54%) gi_115794210 ^(a)SVM: support vectormachines; ^(b)RF: random forest; ^(c)ANN: artificial neural networks;^(d)DA: discriminant analysis; ^(e)Aliphatic index: positivelycorrelated with thermostability; ^(f)Instability Index: <40 isconsidered stable; ^(g)CAMP database - <http://www.camp.bicnirrh.res.in>

The antimicrobial activity of the twelve peptides (Met1-Met12) wastested against S. aureus, E. coli, and Verticillium dahliae. Initialscreening was performed by the radial diffusion assay to confirm theantimicrobial activity. Peptides Met1, Met3, Met4, Met5, Met10, Met11and Met12 were active against E. coli (FIG. 3) and Met3, Met10 and Met11exhibited activity against S. aureus, each at the concentration of 100μg/well. Synergistic effect of the peptides was also tested by mixingequimolar concentrations of peptides, but no significant difference inactivity was seen. Moderate antifungal activity was observed for Met8against Verticillium dahliae (data not shown). Minimal inhibitoryconcentration (MIC) assays were carried out for all samples against E.coli and S. aureus. Met11 was active at the concentration of 95 μM andthe rest of the peptides had a MIC of >190 μM. All peptides werepredicted to have an alpha helical structure except Met12.

By using rational design techniques, we have designed variants ofpeptides by incorporating or replacing various amino acids in theoriginal peptides. Thus, we have designed and modified Chain 100, Chain200 and Chain 300 with tryptophan (W), arginine (R) and lysine (K) atvarious positions to improve antimicrobial activity (FIGS. 4a-c and 5a-c). In some peptides we have also used other amino acids such as Leucine(L), Cysteine (C), Isoleucine (I), Phenylalanine (F) and Alanine (A).For synthesis, 32 variants and 3 original peptides were selected basedon the various parameters such as positive charge, aliphatic index andinstability index. Peptides variants were tested by using radialdiffusion assay against Escherichia coli (ATCC 25922), Staphylococcusaureus (ATCC 25923), Klebsiella pneumoniae (ATCC 10031) and Pseudomonasaeruginosa (ATCC 27853) (FIGS. 6a-c ). The zone of inhibition rangedbetween 10-18 mm for Escherichia coli and Staphylococcus aureus, 6-18 mm& 8-18 mm for Pseudomonas aeruginosa and Klebsiella pneumoniarespectively (Table 2). For Gentamicin it was between 22-24 mm.Antifungal activity was tested for promising antibacterial peptides(Chains 104, 105, 109, 201, 204, 306, 307, 308, 310) against Aspergillusflavus (DSM 1959) and Penicillium chrysogenum (DSM 1075). All thesepeptides has activity at a concentration of 20 μg, Amphotericin B wasused as a positive control at 20 μg (FIG. 7).

Minimum inhibition concentrations (MICs) were tested by microbrothdilution method and Chain 104, 105 and 109 inhibited the growth ofEscherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923),Klebsiella pneumoniae (ATCC 10031) and Pseudomonas aeruginosa (ATCC27853) between 1-8 μg/ml respectively (Tables 3a, 3b and 3c). Themembranolytic activities of the peptides were tested by measuring therelease of hemoglobin from erythrocytes of fresh blood donated byvolunteers. Hemoglobin levels were determined spectrometrically bytaking OD at 540 nm by following the protocol of Schmidtchen et al.,2011. The percent hemolysis ranged between 0.1 to 9% indicating thatmost of these peptides do not have any effect on the host at aconcentration of 128 μg/ml (Table 3).

Twelve peptides, Chain 104, Chain 105, Chain 109, Chain 201, Chain 204,Chain 304, Chain 306, Chain 307, Chain 308, Chain 309, Chain 310 andChain 311 were found to be very active against the four ATCC strains andtherefore, were synthesized as D-form amino acids and tested againstclinical strains of E. coli, K. pneumoniae, E. cloacae, P. aeruginosa,Serratia marcescens, Proteus mirabilis, S. pneumoniae, Acinetobacterbaumannii, A. johnsonii and S. aureus. Clinical strains of yeastsincluding Candida albicans, C. glabrata, C. parapsilosis and C.quillermondiae were also tested with the 12 Chain peptides. The minimuminhibitory concentration (MICs) varied between these peptides and arepresented in Tables 4a, 4b, 4c and 5a, 5b, 5c. Peptides Chain 105, 201and 308 are broad spectrum antimicrobials with MICs between 0.5-32 μg/mlagainst all the bacteria and yeasts tested, except Serratia marcescensand Proteus mirabilis, against which the MICs were between 64-128 μg/ml.Peptide Chain 306 has narrow spectrum activity against E. coli at 1μg/ml and for the rest of the bacterial strains at >32 μg/ml (FIG. 10).

Peptides Chain 104, Chain 105, Chain 201, Chain 308, Chain 310 weretested against antibiotic-sensitive strains of S. aureus RN4220 andmethicillin resistant strains of S. aureus COL (MRSA). Chain peptidescomprising of either L- or D-form amino acids were tested against thesebacteria, and the D-form peptides were found have better activitycompared to L-form (Table 6). Synergistic activity of chain peptidesalong with Ertapenem (4 μg/ml) was tested to know whether it willinfluence the activity against MRSA strains. All the Chain peptides werefound to have antibacterial activity against MRSA strains with MICs atthe range of 0.03 to 4 μg/ml. Chain 310 has the best antibacterialactivity against the MRSA strains with a MIC of 0.06 & 0.03 μg/ml alongwith Ertapenem (4 μg/ml) for both forms of L and D-peptides (Table 6).

Many antimicrobial peptides (AMPs) have broad spectrum antimicrobialactivity against clinical strains of bacteria and fungi. Antimicrobialpeptides form secondary structures by electrostatic interactions withbacterial membranes by a salt-sensitive step. Therefore high saltconcentrations in the human body fluids can deactivate many AMPs and itis essential to develop salt-tolerant AMPs for applications inhealthcare. However, it is well known that physiological condition, pH,temperature and high salt concentrations will influence the activity ofthese peptides. We have evaluated pH, salt- and thermostability of Chain104, 105, 201, 204, 306 and 308 and have found that these peptides arenot hindered by pH or temperature even upto 45° C. for over 14 days(FIGS. 8 & 9). However, there is a 2-fold increase in MIC values ofChain 104, 105, 201, 204, 306 and 308 upon using 200 mM NaClconcentration.

Electron microscopy studies were needed to know the AMP mechanism ofaction on the cells upon treatment with 5× MICs on the cell surface, aswell as the intracellular alterations. AMPs typically cause multiplestresses on the cell membranes even at low concentrations. E. coli has acomplex cell structure with cytoplasmic membrane (7 nm), peptidoglycanlayer (7-8 nm) and an outer membrane (10-15 nm). Antibiotic resistanceof E. coli is due to the presence of inner peptidoglycan along with theouter membrane. Upon incubation with Chain peptides (105, 201, 308),almost all E. coli cells had lost their rod-shaped structural integrity,and accumulation of granular structures, bubble-like structure protrudedfrom cell surface and distortions were seen in cells (FIG. 11). Theinteraction of the Chain peptides with S. aureus lipid bilayer can beseen with an expansion of membrane area. We also observed spreading ofcytoplasmic membrane and spherical mesosomes as intracellular bilayermembranes and mesosome-like structures formed within the cells.

Catheter-associated infections were initiated by the planktonicbacterial cells adherence to the biomaterial surface by colonization andbiofilm formation. Catheters immobilized with Chain 201 or Chain 105posed excellent antimicrobial activity against E. coli and S. aureus. Areduction of more than 70% and 30% of living cells of S. aureus and E.coli was observed, respectively (FIG. 12).

TABLE 2a Antibacterial activity of peptide variants by radial diffusionmethod. Inhibition zone is provided in millimeters for respectivebacterial pathogens. Inhibition zone (mm)* Pseu- EscherichiaStaphylococcus domonas Klebsiella Peptides coli aureus aeruginosapneumoniae Chain200 — — — — Chain201 16 15 15 14 Chain202 — — — —Chain203 16  8  5 10 Chain204 18 15 18 15 Chain205  6  5 — — Chain206 1410 — — Chain207 — — — — Chain208 15 12 10 12 Chain209 14 11 —  8Chain210 13 10 —  6 Gentamycin 22 24 24 22 Test concentration:Gentamycin - 5 μg; Peptides - 50 μg Escherichia coli (ATCC 25922),Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 10031)and Pseudomonas aeruginosa (ATCC 27853) *Values in diameter includingthe wells.

TABLE 2b Antibacterial activity of peptide variants by radial diffusionmethod. Inhibition zone is provided in millimeters for respectivebacterial pathogens. Inhibition zone (mm)* Pseu- EscherichiaStaphylococcus domonas Klebsiella Peptides coli aureus aeruginosapneumoniae Chain200 — — — — Chain201 16 15 15 14 Chain202 — — — —Chain203 16  8  5 10 Chain204 18 15 18 15 Chain205  6  5 — — Chain206 1410 — — Chain207 — — — — Chain208 15 12 10 12 Chain209 14 11 —  8Chain210 13 10 —  6 Gentamycin 22 24 24 22 Test concentration:Gentamycin - 5 μg; Peptides - 50 μg Escherichia coli (ATCC 25922),Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 10031)and Pseudomonas aeruginosa (ATCC 27853) *Values in diameter includingthe wells.

TABLE 2c Antibacterial activity of peptide variants by radial diffusionmethod. Inhibition zone is provided in millimeters for respectivebacterial pathogens. Inhibition zone (mm)* Pseu- EscherichiaStaphylococcus domonas Klebsiella Peptides coli aureus aeruginosapneumoniae Chain300 — — — — Chain301 12 10  5 10 Chain302 12 12 10 —Chain303 12 10  4 — Chain304 14 16 16 11 Chain305 15 12 15 11 Chain30612  8  9 — Chain307 19 20 20 17 Chain308 19 20 20 18 Chain309 11 10 1011 Chain310 17 15 20 16 Chain311 13 14 18 13 Chain312 — — — — Chain313 8  7 14 — Gentamycin 22 24 24 22 Test concentration: Gentamycin - 5 μg;Peptides - 50 μg Escherichia coli (ATCC 25922), Staphylococcus aureus(ATCC 25923), Klebsiella pneumoniae (ATCC 10031) and Pseudomonasaeruginosa (ATCC 27853) *Values in diameter including the wells.

TABLE 3a Minimum inhibitory concentration of different peptide variantstested against 4 bacterial pathogens. Hemolytic activity of the peptideswas tested at 128 μg/ml and percent hemolysis is presented. Minimuminhibitory concentration (μg/ml) Escherichia Staphylococcus Klebsiellacoli aureus pneumoniae Pseudomonas (ATCC (ATCC (ATCC aeruginosa % 25922)25923) 10031) (ATCC 27853) Hemolysis* Chain100 >128 >128 >128 >128 0.4Chain101 >128 >128 >128 >128 4.1 Chain102 >128 >128 >128 >128 5.0Chain103 >128 >128 >128 >128 0.7 Chain104 8 32 16 0.25 0.5 Chain105 2 21 2 0.2 Chain106 >128 >128 >128 64 0.9 Chain107 64 >128 >128 >128 0.1Chain108 >128 >128 >128 >128 0.5 Chain109 8 8 8 16 3.8 Gentamicin 1 0.250.25 0.25 — Tetracycline 8 2 2 32 — *Tested at 128 μg/ml

TABLE 3b Minimum inhibitory concentration of different peptide variantstested against 4 bacterial pathogens. Hemolytic activity of the peptideswas tested at 128 μg/ml and percent hemolysis is presented. Minimuminhibitory concentration (μg/ml) Escherichia coli StaphylococcusKlebsiella Pseudomonas (ATCC aureus pneumoniae aeruginosa % 25922) (ATCC25923) (ATCC 10031) (ATCC 27853) Hemolysis* Chain200 >128 >128 >128 >1280.62 Chain201 2 4 8 8 0.56 Chain202 >128 >128 >128 1 0.56 Chain20332 >128 >128 >128 0.46 Chain204 2 16 32 4 0.55 Chain20564 >128 >128 >128 0.60 Chain206 16 >128 >128 >128 0.29Chain207 >128 >128 >128 >128 0.33 Chain208 32 >128 >128 32 2.01 Chain20964 >128 >128 >128 9.01 Chain210 >128 >128 >128 >128 8.61 Gentamycin 10.25 0.25 0.25 — tetracyclin 8 2 2 32 — *Tested at 128 μg/ml

TABLE 3c Minimum inhibitory concentration of different peptide variantstested against 4 bacterial pathogens. Hemolytic activity of the peptideswas tested at 128 μg/ml and percent hemolysis is presented. Minimuminhibitory concentration (μg/ml) Escherichia coli StaphylococcusKlebsiella Pseudomonas (ATCC aureus pneumoniae aeruginosa % 25922) (ATCC25923) (ATCC 10031) (ATCC 27853) Hemolysis* Chain300 >128 >128 >128 >1286.81 Chain301 64 >128 >128 64 0.24 Chain302 >128 128 >128 16 0.44Chain303 64 >128 >128 >128 0.22 Chain304 16 32 64 8 0.34 Chain305 128 6464 16 0.43 Chain306 8 2 >128 64 0.04 Chain307 8 4 16 1 0.22 Chain308 1 44 2 0.32 Chain309 32 32 64 8 0.09 Chain310 4 8 16 1 3.69 Chain311 16 3232 8 1.18 Chain312 >128 >128 >128 >128 4.87 Chain313 32 >128 >128 645.40 Gentamycin 1 0.25 0.25 0.25 — tetracyclin 8 2 2 32 — *Tested at 128μg/ml

TABLE 4a Minimum inhibitory concentration of peptide variants asdetermined by microdilution method. Bacteria* Strains Chain104 Chain105Chain109 Escherichia coli 1 4 1 16 2 4 4 4 3 4 1 4 4 4 1 4 5 4 2 4Klebsiella pneumoniae 1 8 4 16 2 4 2 8 3 8 4 8 4 16 4 16 5 4 2 8Pseudomonas aeruginosa 1 4 4 16 2 2 4 16 3 4 4 16 4 2 4 16 5 1 4 8Serratia marcescens 1 >128 128 >128 2 >128 128 >128 3 >128 128 >1284 >128 128 >128 5 128 128 64 Proteus mirabilis 1 >128 >128 >128 2 32 3264 3 128 64 64 4 >128 >128 >128 5 >128 >128 >128 Enterobacter cloacae 14 2 4 2 4 2 8 3 8 2 8 4 4 2 8 5 2 1 4 Staphylococcus aureus 1 4 2 2 2 22 4 3 4 2 2 4 2 2 2 5 4 2 2 Streptococcus pneumoniae 1 4 4 16 2 2 2 8 31 2 2 4 4 4 8 5 1 2 8 Acinetobacter baumannii 1 16 4 — 2 8 4 — 3 8 4 — 44 2 — 5 8 4 — Acinetobacter johnsonii 1 8 4 — *The MIC values aredetermined by microdilution method using Muller Hinton broth-cationadjusted in presence of 0.02% BSA and 0.2% acetic acid.

TABLE 4b Minimum inhibitory concentration of peptide variants asdetermined by microdilution method. Bacteria* Strains Chain201 Chain204Escherichia coli 1 16 8 2 1 1 3 1 1 4 1 1 5 1 1 Klebsiella 1 2 8pneumoniae 2 2 4 3 4 16 4 4 4 5 2 2 Pseudomonas 1 4 4 aeruginosa 2 4 163 4 8 4 2 8 5 2 4 Serratia marcescens 1 64 >128 2 64 >128 3 64 128 464 >128 5 128 >128 Proteus mirabilis 1 >128 >128 2 32 32 3 64 644 >128 >128 5 >128 >128 Enterobacter cloacae 1 1 4 2 2 8 3 2 4 4 2 8 5 12 Staphylococcus 1 1 1 aureus 2 1 2 3 1 2 4 1 1 5 1 1 Streptococcus 1 84 pneumoniae 2 4 4 3 2 4 4 4 4 5 4 4 Acinetobacter 1 4 4 baumannii 2 4 43 4 4 4 4 4 5 4 4 Acinetobacter johnsonii *The MIC values are determinedby microdilution method using Muller Hinton broth-cation adjusted inpresence of 0.02% BSA and 0.2% acetic acid.

TABLE 4c Minimum inhibitory concentration of peptide variants asdetermined by microdilution method. Bacteria* Strains Chain304 Chain306Chain307 Chain308 Chain309 Chain310 Chain311 Gentamicin Escherichia coli1 1 1 4 1 0.5 4 2 0.5 2 16 2 4 0.5 32 32 2 0.5 3 16 2 4 1 16 16 1 1 4 322 2 1 32 16 0.5 1 5 32 2 4 1 16 16 1 1 Klebsiella 1 >128 128 8 4 128 648 2 pneumoniae 2 >128 >128 8 4 32 64 8 0.25 3 >128 >128 16 4 >128 128 42 4 >128 64 16 4 32 64 8 2 5 64 64 4 4 128 32 4 4 Pseudomonas 1 32 12816 8 16 8 16 1 aeruginosa 2 64 >128 16 8 32 16 8 0.5 3 64 128 16 4 16 1616 0.5 4 64 >128 8 4 32 8 8 0.5 5 16 128 8 4 8 4 4 0.25 Serratia1 >128 >128 >128 128 >128 >128 >128 1 marcescens 2 >128 >128 >128128 >128 >128 >128 2 3 >128 >128 128 64 >128 >128 >128 14 >128 >128 >128 >128 >128 >128 >128 1 5 >128 >128 >128128 >128 >128 >128 1 Proteus mirabilis1 >128 >128 >128 >128 >128 >128 >128 2 2 >128 >128 128 32 >128 >128 1282 3 >128 >128 128 128 >128 >128 128 >1284 >128 >128 >128 >128 >128 >128 >128 85 >128 >128 >128 >128 >128 >128 >128 >128 Enterobacter 1 64 64 4 2 >128128 4 0.25 cloacae 2 64 64 8 2 128 64 4 0.25 3 128 32 4 2 64 16 4 0.25 4128 16 4 2 64 8 4 0.25 5 32 16 4 2 32 16 2 0.25 Staphylococcus 1 32 16 21 8 8 1 0.5 aureus 2 32 32 2 2 16 16 1 0.5 3 16 32 2 1 8 8 1 0.5 4 16 162 2 8 8 1 0.5 5 16 8 2 2 8 4 1 0.5 Streptococcus 1 128 64 4 8 64 128 162 pneumoniae 2 128 64 4 4 64 128 8 2 3 128 64 4 8 64 128 16 2 4 128 1282 2 64 128 4 2 5 64 64 2 4 64 64 8 1 Acinetobacter 1 — 16 — 4 — — — 0.5baumannii 2 — 32 — 4 — — — 0.25 3 — 16 — 4 — — — 0.5 4 — 16 — 2 — — —0.5 5 — 8 — 2 — — — 0.25 Acinetobacter 1 — 16 — 4 — — — 0.25 johnsonii*The MIC values are determined by microdilution method using MullerHinton broth-cation adjusted in presence of 0.02% BSA and 0.2% aceticacid.

TABLE 5a Minimum inhibitory concentration of peptide variants testedagainst clinical yeast strains Fungi Strains Chain104D Chain105DChain109D Candida albicans 1 8 32 128 2 8 16 128 3 8 32 128 4 8 32 128 58 32 128 C. glabrata 1 8 16 128 2 8 16 128 3 8 8 16 4 16 32 64 C.parapsilosis 1 4 8 128 2 4 8 128 3 4 8 128 4 2 4 8 C. quillermondiae 1 48 128

TABLE 5b Minimum inhibitory concentration of peptide variants testedagainst clinical yeast strains Fungi Strains Chain201D Chain204D Candida1 16 8 albicans 2 8 8 3 8 8 4 16 8 5 8 8 C. glabrata 1 8 4 2 8 8 3 8 324 64 32 C. parapsilosis 1 8 4 2 8 4 3 8 8 4 8 8 C. quillermondiae 1 8 4

TABLE 5c Minimum inhibitory concentration of peptide variants testedagainst clinical yeast strains Fungi Strains Chain304D Chain306DChain307D Chain308D Chain309D Chain310D Chain311D Amphotericin B Candidaalbicans 1 16 16 16 16 32 16 8 2 2 16 8 16 16 32 16 8 0.5 3 16 8 16 1616 16 8 2 4 16 16 16 16 16 16 16 2 5 32 16 16 16 32 16 16 2 C. glabrata1 16 8 8 16 32 16 16 4 2 16 8 16 16 64 16 16 4 3 8 16 8 16 4 128 8 2 464 32 16 16 128 128 64 4 C. parapsilosis 1 64 8 16 8 64 64 16 2 2 64 168 8 64 64 8 2 3 32 16 8 8 32 64 8 2 4 16 4 8 4 16 16 8 2 C.quillermondiae 1 16 4 4 4 8 8 4 2

TABLE 6 Minimum inhibitory concentration (MICs) of peptide variantsagainst antibiotic resistant and sensitive strains of S. aureus andsynergistic activity with Ertapenem. S. aureus COL + S. aureus S. aureus4 μg/ml Compound RN4220 COL (MRSA) Ertapenem 104 >64 μg/ml >64 μg/ml0.25 μg/ml 104 D 64 μg/ml 32 μg/ml 4 μg/ml 105 >64 μg/ml >64 μg/ml 0.5μg/ml 105 D 16 μg/ml 8 μg/ml 2 μg/ml 201 64 μg/ml 64 μg/ml 0.5 μg/ml 201D 2 μg/ml 2 μg/ml 1 μg/ml 308 >64 μg/ml >64 μg/ml 0.5 μg/ml 308 D 4μg/ml 4 μg/ml 1 μg/ml 310 >64 μg/ml >64 μg/ml 0.06 μg/ml 310 D >64μg/ml >64 μg/ml <0.03 μg/ml *D—D-Amino acids.

REFERENCES

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The invention claimed is:
 1. An antimicrobial peptide, comprising anyone of synthetically prepared SEQ ID NOs: 55, 58, 59 and 61 to 65peptide or said antimicrobial peptide defined by a peptide sequencehaving at least 90 identity to any one of SEQ ID NOs: 55, 58, 59 and 61to
 65. 2. The peptide of claim 1, wherein said peptide is modified inorder to increase antimicrobial activity.
 3. The peptide of claim 1,comprising any one of SEQ ID NOs: 55, 58, 59 and 61 to
 65. 4. Thepeptide according to claim 1, having an activity against fungi or yeast.5. The peptide according to claim 1, having an activity againstbacteria.
 6. A peptide as defined in claim 1 for use in therapy, as anantimicrobial agent.
 7. A method of killing or inhibiting growth ofmicrobes, comprising a step of treating said microbes with a peptideaccording claim
 1. 8. The method according to claim 7, wherein thepeptide exhibits an activity against bacteria or fungi or both bacteriaand fungi.